Aspects of the present invention relate generally to improved methods of blocking DNA polymerase activity with oligonucleotide aptamers at low reaction temperatures, and restoring the enzyme activity upon raising the reaction temperature (e.g., hot-start methods).
DNA polymerases are enzymes used for synthesis of DNA strands by primer extension, wherein the polymerase-catalyzed DNA synthesis may be initiated by oligonucleotide primers hybridized to a complementary template DNA. Initiating DNA synthesis from this template-hybridized primer, DNA polymerases create complementary DNA strands in the presence of corresponding nucleotide 5′-triphosphates. Sequence specificity of nucleotide polymerization, when the oligonucleotide primers bind exclusively to the desired sites and nowhere else, is an important requirement in many applications wherein DNA synthesis is used. However, the efficiency and fidelity of DNA synthesis can be reduced when primers hybridize to non-complementary DNAs, leading to synthesis of incorrect DNA sequences.
Many so-called ‘Hot Start’ methods have been developed to avoid incorrect primer extension products (e.g., see Paul, N., et al. (2010), for review). One of the most common techniques is based on use of oligonucleotide aptamers (Jayasena S. D., 1999). Aptamers offer a number of advantages over other reported methods. Using a method of molecular evolution (SELEX), they can be quickly engineered in a test tube and then readily and inexpensively manufactured by chemical synthesis. Ideally, an aptamer should: (i) completely block DNA polymerase at low temperatures, and (ii) provide no blockage effect at the desired elevated reaction temperature. Unfortunately, this is very difficult, if not impossible to achieve, and the aptamer structure usually represents a compromise between these two key requirements. New methods, therefore, are needed to improve control of aptamer activity in reaction mixtures containing DNA polymerases.
Particular aspects provide methods for activating an aptamer-inactivated DNA polymerase, comprising: providing a reaction mixture suitable for DNA synthesis, the reaction mixture comprising (i) a DNA polymerase, (ii) a uracil-DNA glycosylase enzymatic activity, and (iii) a DNA polymerase-binding oligonucleotide aptamer that comprises a hairpin structure having a stem sequence portion and a loop sequence portion, wherein the loop sequence portion comprises one or more deoxyuridine nucleotide(s) modifiable by the uracil-DNA glycosylase enzymatic activity, and the aptamer is present in an amount sufficient to inhibit DNA synthesis activity of the DNA polymerase in the reaction mixture; and modifying the aptamer by the uracil-DNA glycosylase enzymatic activity to form a modified aptamer having less or no inhibitory effect on the DNA polymerase, thereby activating or enhancing the DNA synthesis activity of the DNA polymerase, to start and/or increase DNA synthesis in the reaction mixture. In the methods, for example, modifying the aptamer may be facilitated by use of a reaction temperature that facilitates the DNA polymerase activity and/or the uracil-DNA glycosylase enzymatic activity. In the methods, modifying the aptamer may be facilitated by increasing the temperature of the reaction mixture from a first temperature to a second temperature that activates or more strongly facilitates the uracil-DNA glycosylase enzymatic activity. In the methods, providing the reaction mixture may comprise dissolving a dried form of at least one of the (i) DNA polymerase, (ii) oligonucleotide aptamer, and (iii) uracil-DNA glycosylase enzymatic activity, into an aqueous solution. In the methods, the DNA synthesis may result in DNA amplification in the reaction mixture (e.g., wherein the DNA amplification is an isothermal amplification reaction, and/or wherein the DNA amplification is PCR). The methods may comprise detecting a presence of a target DNA in the reaction mixture, and/or measuring an amount of a target DNA in the reaction mixture. In the methods, the oligonucleotide aptamer may be circular. In the methods, the uracil-DNA glycosylase enzymatic activity may be effective to modify the oligonucleotide aptamer by generating at least one abasic site within the loop sequence portion thereof. In the methods, the uracil-DNA glycosylase may be or comprise Afu Uracil-DNA Glycosylase. In the methods, the loop sequence portion may be or comprise a nucleotide sequence 5′-TTCTTAGCGTTT-3′ (SEQ ID NO:23) wherein one or more thymidine nucleotides at positions 1, 2, 10, 11, and 12 of the SEQ ID NO:23 sequence are substituted by one or more deoxyuridine nucleotides.
Additional aspects provide kits for activating an aptamer-inactivated DNA polymerase, comprising: a uracil-DNA glycosylase enzymatic activity; and a DNA polymerase-binding oligonucleotide aptamer sequence that is capable of forming a hairpin structure having a stem sequence portion and a loop sequence portion, wherein the loop sequence portion comprises one or more deoxyuridine nucleotides modifiable by the uracil-DNA glycosylase enzymatic activity. In the kits, the loop sequence portion may be or comprise a nucleotide sequence 5′-TTCTTAGCGTTT-3′ (SEQ ID NO:23) wherein one or more thymidine nucleotides at positions 1, 2, 10, 11, and 12 of the SEQ ID NO:23 sequence are substituted by one or more deoxyuridine nucleotides. In the kits, the oligonucleotide aptamer may be a circular molecule. In the kits, the uracil-DNA glycosylase enzymatic activity may be effective to modify the oligonucleotide aptamer by generating at least one abasic site within the loop sequence portion. In the kits, the uracil-DNA glycosylase may be or comprise Afu Uracil-DNA Glycosylase.
Further aspects provide reaction mixtures for use in a method of DNA synthesis, which reaction mixture comprises: (i) a DNA polymerase, and (ii) a DNA polymerase-binding oligonucleotide aptamer that comprises a hairpin structure having a stem sequence portion and a loop sequence portion, wherein the loop sequence portion comprises one or more deoxyuridine nucleotides modifiable by a uracil-DNA glycosylase enzymatic activity, and the aptamer is present in an amount sufficient to inhibit DNA synthesis activity of the DNA polymerase in the reaction mixture. The reaction mixtures may further comprise (iii) a uracil-DNA glycosylase enzymatic activity sufficient, under suitable conditions, to modify the oligonucleotide aptamer to reduce or eliminate binding of the oligonucleotide aptamer to the DNA polymerase, thereby activating or enhancing the DNA synthesis activity of the DNA polymerase. In the reaction mixtures, at least one of the DNA polymerase activity and/or the uracil-DNA glycosylase enzymatic activity may be temperature dependent. In the reaction mixtures, the uracil-DNA glycosylase enzymatic activity may increase with increasing temperature of the reaction mixture from a first temperature to a second temperature that activates or more strongly facilitates the uracil-DNA glycosylase enzymatic activity. In the reaction mixtures, the DNA polymerase, and/or oligonucleotide aptamer, and/or uracil-DNA glycosylase enzymatic activity may be present in the reaction mixture in a dried state. In the reaction mixtures, the loop sequence portion may be or comprise a nucleotide sequence 5′-TTCTTAGCGTFT-3′ (SEQ ID NO:23) wherein one or more thymidine nucleotides at positions 1, 2, 10, 11, and 12 of the SEQ ID NO:23 sequence are substituted by one or more deoxyuridine nucleotides. In the reaction mixtures, the oligonucleotide aptamer may be a circular molecule. In the reaction mixtures, the uracil-DNA glycosylase enzymatic activity may be effective to modify the oligonucleotide aptamer by generating at least one abasic site within the loop sequence portion. In the reaction mixtures, the uracil-DNA glycosylase may be or comprise Afu Uracil-DNA Glycosylase. The reaction mixtures may further comprise one or more of dATP, dCTP, dGTP, and/or dTTP, and/or Mg2+ ion.
Yet further aspects provide an oligonucleotide aptamer, comprising a DNA polymerase-binding nucleic acid sequence that is capable of forming a hairpin structure having a stem sequence portion and a loop sequence portion, wherein the loop sequence portion is or comprises a nucleotide sequence 5′-TTCTTAGCGTTT-3′ (SEQ ID NO:23), wherein one or more thymidine nucleotides at positions 1, 2, 10, 11, and 12 of the SEQ ID NO:23 sequence are substituted by one or more deoxyuridine nucleotides modifiable by a uracil-DNA glycosylase enzymatic activity (e.g., by Afu Uracil-DNA Glycosylase). The oligonucleotide aptamers may be circular molecules. The circular oligonucleotide aptamers may comprises a duplex stem sequence portion positioned between two loop sequence portions. In the circular oligonucleotide aptamers comprising a duplex stem sequence portion positioned between two loop sequence portions, the two loop sequence portions may be the same or different sequences. The oligonucleotide aptamers may be in combination with a DNA polymerase.